Methods for immobilizing a ligand onto a carrier have been studied in large numbers for researches in an immobilized enzyme, and the like, and also industrially utilized for producing an adsorbent and the like. Representative examples of the methods are the following methods:
(1) a method for generating an imido carbonate by a cyanogen bromide activating method and successively reacting it with an amino group of a ligand, PA1 (2) a method known as an acid azide derivative method, which comprises esterifing a carboxyl group on a carrier, converting the resulting ester into a hydrazide, successively converting the resulting hydrazide into an azide, and finally replacing the resulting azide group with an amino group of a ligand, PA1 (3) a method known as a diazo method, which comprises generating a diazonium salt on a carrier and making it react with an amino group of a ligand, PA1 (4) a method known as a condensing reagent method, which comprises condensing an amino group or carboxyl group on a carrier with a carboxyl group or amino group of a ligand with a condensing reagent, PA1 (5) a method known as an alkylating method, which comprises introducing an acetyl bromide group or 4,6-dichloro-s-triazinyl group onto a carrier and making it react with an amino group of a ligand, PA1 (6) a method known as a carrier-crosslinking method, which comprises crosslinking an amino group on a carrier and an amino group of a ligand with glutaraldehyde and reducing the resulting compound, and the like (Atsuo Tanaka, Takuo Kawamoto, Gendai Kagaku, pp. 24-30, July 1992). A method for immobilizing a ligand onto a solvent-insoluble carrier having an aldehyde group is based on the method (6) mentioned above. And this method comprises reacting a ligand having an amino group to generate a Schiff base and successively reducing it (a reductive amination). PA1 (a) In case there are plural functional groups to be used for immobilization in a ligand, it is extremely difficult to immobilize the ligand with a functional group existing at a prescribed position of the ligand, because there are plural immobilization points. Furthermore, it is not preferable that there are many functional groups in a ligand, because the possibility that many bonds are formed at many points in the ligand becomes high. Also, in case the number of functional groups in the above-mentioned ligand is reduced in order to avoid forming bonds at multi-points, a reaction yield becomes low. The methods corresponding to these are the above-mentioned methods (1), (2), (3), (4), (5) and (6). PA1 (b) There are many competitive side reactions, and a reaction yield is low. The methods corresponding to these, are the above-mentioned methods (2), (3), (4) and (5). PA1 (1) the carrier of the porous gel is hardly destroyed or generates finely divided particles by the operation of stirring, and the like because of a relatively high mechanical strength and toughness. Since the carrier is neither compacted nor clogged up when a column is charged therewith and a liquid is passed through the column at a high flow rate, a liquid can be passed through the column at a high flow rate. Further, the pore structure hardly changes by high-pressure steam sterilization; PA1 (2) the carrier is hydrophilic since the gel is constituted by crystalline cellulose or crosslinked-cellulose. There exist many OH groups available for bonding a ligand, and non-specific adsorption is also a little; PA1 (3) the gel has relatively high strength, even if volume of porosity thereof is enlarged. Thus, capacity of adsorption thereof which is not inferior to that of a soft gel is obtained; and the like.
Outlines of the above-mentioned immobilizing methods (1)-(6) are shown by the following reaction formulas. In the following chemical reaction formulas, Z is a carrier and E is an enzyme. ##STR2##
However, these methods have the following defects.
In other words, these methods have considerable limits in immobilizing a ligand at a prescribed position thereof onto a solvent-insoluble carrier. Especially in case a peptide or protein is used as a ligand, it is impossible to immobilize a ligand with a prescribed amino group onto the carrier, because a peptide or protein often has amino groups in a side chain (in other words, there exist plural amino groups in one molecule).
On the other hand, it has been known as a reaction of organic synthesis chemistry for a long time that a thiazolidine structure is formed when an aldehyde compound and a derivative of aminoethanethiol (HSCH.sub.2 CH.sub.2 NH.sub.2) are reacted in the presence of an acidic or basic catalyst in an aqueous solution (Schmolka IR, J. Amer. Chem. Soc., 79, p.4716(1957). In late years, this classical reaction has been studied for applying it to bond formation between peptides, and it has been reported recently that peptides can be bonded each other efficiently by this reaction (C. F. Liu, J. P. Tam, Proc. Natl. Acad. Sci. USA, 91, p.6584(1994), C. Rao and J. P. Tam, J. Am. Chem. Soc.), 116, p.6975(1994)).
This method introduces an aldehyde group into the C-terminus of one of two kinds of peptides and has it react with the other peptide (N-terminal cysteine), and the reaction is performed in an aqueous solution wherein both reactants are dissolved in water. In other words, it can be said that such a reaction is known as a reaction only in a uniform solution.
Generally, a reaction between a solid phase and a liquid phase is extremely difficult to proceed. For example, when a dehydration condensation is performed between a terminal amino group (--NH.sub.2) of a hydrophilic compound M having an amino group, for example, a peptide, an amino acid, a protein or a derivative thereof, and a terminal carboxyl group (--COOH) of a hydrophilic compound N having a carboxyl group, for example, a peptide, an amino acid, a protein or a derivative thereof, using DCC (dicyclohexylcarbodiimide), the fact is well-known that a reaction between a carboxyl group of the compound N and DCC does not proceed and a dehydration condensation does not occur, because DCC is insoluble in water (DCC exists as a solid) in an aqueous solution. In other words, it seems quite difficult to apply the above-mentioned reaction reported by C. F. Liu et al. and by C. Rao et al. to a reaction between a solid phase and a liquid phase. As a matter of fact, there are still no example that the above-mentioned reaction is applied to a two-phase reaction of solid-liquid phases.
An adsorbent is widely used in fields such as experimentation, industry, medical treatment and diagnosis purposes of analysis, separation, purification and removal. Fundamental components of an adsorbent consist of a so-called carrier, which is a solid insoluble in an elution solvent, and a so-called ligand, which is a compound having a high affinity for an objected substance.
A method, by which a ligand is immobilized specifically at a prescribed position of a ligand with a high reaction efficiency to form a stable bond, is now eagerly demanded as a method for immobilizing a ligand onto a solvent-insoluble carrier. However, there is not such a method at present. After the present inventors knew well the high degree of difficulty and the strong demand as to establishment of such a method, they started tackling this problem.
Therefore, an object of the present invention is to provide a method for immobilizing a ligand or a compound having a ligand bonded thereto onto a solvent-insoluble carrier, which comprises efficiently and specifically reacting the ligand or the compound with the carrier at a prescribed position to form a stable bond to the carrier.